Hepatitis B Virus Entry into Cells.

Hepatitis B virus (HBV), an enveloped partially double-stranded DNA virus, is a widespread human pathogen responsible for more than 250 million chronic infections worldwide. Current therapeutic strategies cannot eradicate HBV due to the persistence of the viral genome in a special DNA structure (covalently closed circular DNA, cccDNA). The identification of sodium taurocholate co-transporting polypeptide (NTCP) as an entry receptor for both HBV and its satellite virus hepatitis delta virus (HDV) has led to great advances in our understanding of the life cycle of HBV, including the early steps of infection in particular. However, the mechanisms of HBV internalization and the host factors involved in this uptake remain unclear. Improvements in our understanding of HBV entry would facilitate the design of new therapeutic approaches targeting this stage and preventing the de novo infection of naïve hepatocytes. In this review, we provide an overview of current knowledge about the process of HBV internalization into cells.

Copper as the most likely pathogenic divergence factor between lung fibrosis and emphysema.

Although fibrosis and emphysema are in many ways on opposite ends of the pulmonary parenchymal disease spectrum, they seem to share common pathomechanistic steps. This is illustrated by the coexistence of both entities in lungs of individuals with combined pulmonary fibrosis and emphysema. Macroproteins elastin and collagen are major constituents of the pulmonary extracellular matrix. The prevailing paradigm states that emphysema is caused by an imbalance between destructive proteolytic and protective antiproteolytic enzymes leading to accelerated degradation of elastin fibers in the lungs. Rates of elastin breakdown, however, are equally enhanced in patients with idiopathic pulmonary fibrosis (IPF) and emphysema. Excessive accumulation of collagen is a hallmark of IPF. Surprisingly, collagen levels in the lung parenchyma of patients with emphysema are also higher than in controls. The concentration of elastin fibers is elevated in fibrotic lungs, despite accelerated elastinolysis, suggesting that elastin repair is also enhanced in IPF. Since elastin concentrations are reduced in emphysematous lungs, the factor of divergence between emphysema and fibrosis seems to be the degree of elastin repair. Multiple elastin repair steps can be deduced of which tropoelastin synthesis and crosslinking of tropoelastin polymers by the copper dependent enzyme lysyl oxidase seem to be the most important ones. We suspect that the distinction in the pathogeneses of lung fibrosis and emphysema depends on the local availability of copper to activate sufficient lysyl oxidase for elastin crosslinking, and suggest assessing the effects of inhalation therapy with copper plus heparin in emphysema and heparin monotherapy in IPF.

Heparin, Heparan Sulphate and the TGF-ß Cytokine Superfamily.

Of the circa 40 cytokines of the TGF-ß superfamily, around a third are currently known to bind to heparin and heparan sulphate. This includes TGF-ß1, TGF-ß2, certain bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs), as well as GDNF and two of its close homologues. Experimental studies of their heparin/HS binding sites reveal a diversity of locations around the shared cystine-knot protein fold. The activities of the TGF-ß cytokines in controlling proliferation, differentiation and survival in a range of cell types are in part regulated by a number of specific, secreted BMP antagonist proteins. These vary in structure but seven belong to the CAN or DAN family, which shares the TGF-ß type cystine-knot domain. Other antagonists are more distant members of the TGF-ß superfamily. It is emerging that the majority, but not all, of the antagonists are also heparin binding proteins. Any future exploitation of the TGF-ß cytokines in the therapy of chronic diseases will need to fully consider their interactions with glycosaminoglycans and the implications of this in terms of their bioavailability and biological activity.

Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity.

BACKGROUND & AIMS: Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles. METHODS: ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted. RESULTS: Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs). CONCLUSIONS: Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of the availability of ApoE likely influences HCV infectivity, antibody escape and transmission.

Endothelial glycocalyx degradation induces endogenous heparinization in patients with severe injury and early traumatic coagulopathy.

BACKGROUND: There is emerging evidence that early trauma-induced coagulopathy (TIC) is mechanistically linked to disruption of the vascular endothelium and its glycocalyx, assessed by thrombomodulin and syndecan 1, respectively. This study evaluated if degradation of the endothelial glycocalyx and ensuing release of its heparin-like substances induce autoheparinization and thereby contributes to TIC. METHODS: Prospective observational study of 77 trauma patients admitted to a Level I trauma center having blood sampled at admission. Data on demography, hematology, Injury Severity Score, transfusion requirements, 30-day mortality, and thrombelastography (TEG, concurrent kaolin-activated/kaolin-heparinase-activated) were recorded. Retrospective analysis of plasma/serum for biomarkers reflecting endothelial glycocalyx and cell damage (syndecan 1, thrombomodulin), tissue injury (histone-complexed DNA fragments), sympathoadrenal activation (adrenaline, noradrenaline), coagulation activation/anticoagulation (prothrombin fragment 1+2, fibrinogen, von Willebrand factor, factor XIII, antithrombin, protein C, activated protein C, tissue factor pathway inhibitor), fibrinolysis (tissue-type plasminogen activator, plasminogen activator inhibitor 1) and inflammation (interleukin 6, terminal complement complex). Stratification of patients was according to the degree of TEG-measured heparinization. RESULTS: Four patients (5.2%) displayed evidence of high-degree autoheparinization, and these patients had higher Injury Severity Score (median [interquartile range], 31 [26-37] vs. 17 [10-26]), increased glucose (median, 13.6 vs. 8.0 mmol/L), and lower hemoglobin level (median, 5.8 vs. 8.4 mmol/L) and received more transfusions during the first 1 hour (median, 5 vs. 0) and 24 hours (median, 10 vs. 0) (all p < 0.05). Importantly, patients with autoheparinization had fourfold higher syndecan 1 levels (median [interquartile range], 116 ng/mL [78-140 ng/mL] vs. 31 ng/mL [18-49 ng/mL]), and they had higher international normalized ratio (median, 1.4 vs. 1.1), thrombomodulin (median, 4.1 vs. 1.7 ng/mL) and interleukin 6 (median, 129 vs. 71 pg/mL) but lower protein C (85% vs. 109%) (all p < 0.05), indicating profound endothelial damage, coagulopathy and inflammation. CONCLUSION: Five percent of the patients with trauma in the present study had evidence of acute endogenous coagulopathy with autoheparinization by TEG, which appeared mechanistically linked to endothelial glycocalyx degradation. Acute endogenous autoheparinization may contribute to TIC. LEVEL OF EVIDENCE: Prognostic study, level III.

Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.

OBJECTIVE: This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. STUDY DESIGN: Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. RESULTS: Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. CONCLUSION: These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.

[Regulatory role of heparin compounds with low molecular blood ligands in plasma and thrombocyte hemostasis].

Analysis of the literature and our own research on the physiological effects of complex compounds of heparin with low molecular ligands (amino acids, regulatory peptides) is presented. It is proved that anticoagulative effects in blood flow were conditioned by the interaction of heparin with glioproline, immunopeptides, and other low molecular substances with formation of complex compounds. The presence of structural regions of binding of heparin and other components is established. It is indicated that in the blood of animals heparin complexes with low molecular ligands possess protective anticoagulative and antithrombotic effects. We made an attempt to reveal the possible mechanism of anticoagulative-fibrinolytic and antithrombotic action of complex compounds of heparin in the organism.

Characterization of the envelope glycoproteins associated with infectious hepatitis C virus.

Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly.

LTBP-2 has multiple heparin/heparan sulfate binding sites.

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca(2+) ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca(2+) dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG-LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.

Heparin is a promising agent for the treatment of endometriosis-associated fibrosis.

OBJECTIVE: To assess the use of heparin for the medical treatment of endometriosis-associated fibrosis. DESIGN: The effects of heparin on the endometriotic stromal cells (ECSCs)-mediated contractility were investigated. SETTING: Research laboratory at a medical school. PATIENT(S): Endometriotic tissues from nine patients were used. INTERVENTION(S): Endometriotic stromal cells were cultured three dimensionally in the presence of heparin. MAIN OUTCOME MEASURE(S): The contractility of ECSCs was assessed by collagen gel contraction assay. Heparin-induced morphological changes of ECSCs were evaluated by laser scanning microscopy. The expression of contractility-related molecules in ECSCs was examined by Western blot analysis. RESULT(S): In the presence of 10% fetal bovine serum, treated ECSCs showed significant collagen gel contractility (75.9% decrease in surface area after 48 hour vs. 0 hour controls). Endometriotic stromal cell-mediated gel contraction was significantly attenuated in the presence of heparin in a dose-dependent manner (55.7% reduction of the gel contraction at a concentration of 100 microg/mL of heparin sodium versus untreated controls after 48 hours). Heparin suppressed the ECSC attachment to collagen fibers. The expression of alpha-smooth muscle actin, Ras homology (Rho) A, Rho-associated coiled-coil-forming protein kinase (ROCK)-I, and ROCK-II was down-regulated by heparin administration. CONCLUSION(S): The present study suggests that heparin is a promising agent for the treatment of endometriosis-associated fibrosis. The inhibition of myofibroblastic differentiation, the attenuation of attachment to collagen fibers, and the suppression of Rho-ROCK-mediated pathway activation in ECSCs are involved in the action mechanisms of heparin.

Heparin and low-molecular-weight heparins modulate the decidualization of human endometrial stromal cells.

OBJECTIVE: To examine the impact of unfractionated heparin and low-molecular-weight heparins (LMWHs) on the decidualization of human endometrial stromal cells (ESCs) in vitro. DESIGN: In vitro experiment. SETTING: Research laboratory at a medical university center. PATIENT(S): Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S): The ESCs were isolated from hysterectomy specimens, decidualized in vitro using progesterone and 17beta-estradiol, and incubated with unfractionated heparin and three different LMWHs. MAIN OUTCOME MEASURE(S): Insulin-like growth factor-binding protein (IGFBP) 1, PRL, and insulin-like growth factor (IGF) I were measured using ELISA and real-time reverse-transcription polymerase chain reaction. Cell viability was determined by a fluorometric assay. Intracellular cyclic adenosine 3',5'-monophosphate (cAMP) was measured using a luminescent assay. RESULT(S): Heparin dose- and time-dependently delayed the production of IGFBP-1 and amplified the levels of PRL and IGF-I in ESCs during decidualization in vitro. Similar effects were seen under the influence of the three different LMWHs. Intracellular cAMP was increased in decidualizing ESCs under the influence of heparin and LMWHs. CONCLUSION(S): Unfractionated heparin as well as LMWHs are able to modulate the decidualization of human ESCs in vitro and therefore might be useful to control endometrial differentiation and receptivity in assisted reproduction.

Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Abeta40 (DAbeta(1-40)) are modulated by sulfate moieties of heparin.

Glycosaminoglycans (GAGs), in particular as part of heparan sulfate proteoglycans, are associated with cerebral amyloid angiopathy (CAA). Similarly, GAGs are also associated with the severe CAA found in patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), where the amyloid beta (Abeta) peptide contains the Dutch mutation (DAbeta(1-40)). This suggests a role for GAGs in vascular Abeta aggregation. It was the aim of this study to investigate the effect of different GAGs (heparin, chondroitin sulfate, heparan sulfate), the macromolecule dextran sulfate and, using desulfated heparins, the role of GAG sulfate moieties on the in vitro aggregation of CAA-associated DAbeta(1-40) and on DAbeta(1-40)-induced toxicity of cultured cerebrovascular cells. We also aimed to study the in vivo distribution of various sulfated heparan sulfate GAG epitopes in CAA. Of all GAGs tested, heparin was the strongest inducer of aggregation of DAbeta(1-40) in the different aggregation assays, with both heparin and heparan sulfate reducing Abeta-induced cellular toxicity. Furthermore, (partial) removal of the sulfate moieties of heparin partially abolished the effects of heparin on aggregation and cellular toxicity, suggesting an essential role for the sulfate moieties in heparin. Finally, we demonstrated the in vivo association of sulfated heparan sulfate (HS) GAGs with CAA. We conclude that sulfate moieties within GAGs, like heparin and HS, have an important role in Abeta aggregation in CAA and in Abeta-mediated toxicity of cerebrovascular cells.

Heparin, heparan sulfate and heparanase in inflammatory reactions.

Heparan sulfate (HS) proteoglycans at the cell surface and in the extracellular matrix of most animal tissues are essential in development and homeostasis, and are implicated in disease processes. Emerging evidence demonstrates the important roles of HS in inflammatory reactions, particularly in the regulation of leukocyte extravasation. Heparin, a classical anticoagulant, exhibits anti-inflammatory effects in animal models and in the clinic, presumably through interference with the functions of HS, as both polysaccharides share a high similarity in molecular structure. Apart of regulation during biosynthesis, the structures of HS and heparin are significantly modulated by heparanase, an endoglycosidase that is upregulated in a number of inflammatory conditions. Exploring the physiological roles of HS and heparin and the mode of heparanase action in modulating their functions during inflammation responses is of importance for future studies.

The heparin binding motif of endostatin mediates its interaction with receptor nucleolin.

Endostatin is a potent angiogenesis inhibitor with heparin-dependent activities. Nucleolin, a novel functional receptor of endostatin, mediates both the internalization to endothelial cells and the antiangiogenic activity of endostatin. To define the exact role of the heparin binding motif in mediating the interaction between endostatin and its receptor nucleolin, up to six arginine residues (R155, R158, R184, R270, R193, and R194) located in the heparin binding motif of endostatin were substituted by alanine to make double, quadruple, or hexad point mutations, respectively. Contributions of the heparin binding motif to both the interaction with nucleolin and the biological activities of endostatin were investigated from in vitro to in vivo. Here we show that Arg to Ala point mutagenesis of the heparin binding motif does not interrupt the folding of endostatin but significantly impairs the interaction between endostatin and nucleolin. Double and quadruple mutants showed significantly decreased internalization to endothelial cells and antitumor activities, while the hexad Arg to Ala mutant completely lost its interaction with nucleolin and biological functions. Taken together, the present study demonstrates that the arginine clusters in the heparin binding motif of endostatin significantly contribute to its interaction with receptor nucleolin and mediate the antiangiogenic and antitumor activities of endostatin.

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

BACKGROUND AND OBJECTIVE: A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. MATERIAL AND METHODS: Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. RESULTS: Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. CONCLUSION: These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

Bevacizumab immune complexes activate platelets and induce thrombosis in FCGR2A transgenic mice.

BACKGROUND: Treatment with Bevacizumab has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. OBJECTIVE: We investigated whether thrombosis might be the result of platelet activation mediated via the FcgammaRIIa (IgG) receptor - which is not present on mouse platelets - and aimed to identify the functional roles of heparin and platelet surface localization in Bev-induced FcgammaRIIa activation. METHODS AND RESULTS: We found that Bev immune complexes (IC) activate platelets via FcgammaRIIa, and therefore attempted to reproduce this finding in vivo using FcgammaRIIa (hFcR) transgenic mice. Bev IC were shown to be thrombotic in hFcR mice in the presence of heparin. This activity required the heparin-binding domain of Bev's target, vascular endothelial growth factor (VEGF). Heparin promoted Bev IC deposition on to platelets in a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia. When sub-active amounts of ADP or thrombin were used to prime platelets (simulating hypercoagulability in patients), Bev IC-induced dense granule release was significantly potentiated, and much lower (sub-therapeutic) heparin concentrations were sufficient for Bev IC-induced platelet aggregation. CONCLUSIONS: The prevailing rationale for thrombosis in Bev therapy is that VEGF blockade leads to vascular inflammation and clotting. However, we conclude that Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of the platelet FcgammaRIIa receptor, and that this provides a better explanation for the thrombotic events observed in vivo.

Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation.

Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0.1 microg/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.

The heparanome and regulation of cell function: structures, functions and challenges.

The cell-extracellular matrix interface is a crowded space whose structure is dependent on macromolecular assemblies that are dynamic in time, molecular composition and location. Signals travel from one cell to another (or to the same cell) by the regulated assembly/disassembly of molecular complexes. These signals can evoke relatively simple biological responses such cell proliferation and migration, but once integrated, they guide cell fate in complex biological phenomena such as embryonic development and organism homeostasis. Heparan sulfate proteoglycans are ubiquitous components of this space and important actors of these processes in all tissue-organized life forms. A key feature of heparan sulfate is its size, 40 nm to 160 nm, which enables it to integrate self-assembling macromolecular structures over substantial length scales. What is the structure of heparan sulfate? Why do we think heparan sulfate is so important? How do we try to explain its activity? What do we know about its interactions? These questions together with a final look to the future are the "menu" of this review.

Linkage-dependent contribution of repeat peptides to self-aggregation of three- or four-repeat microtubule-binding domains in tau protein.

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism for the tau protein, it is still unclear how it is transformed from a normal structure in a neuron. To examine the linkage-dependent contribution of each repeat peptide (R1-R4) to filament formation of the three- or four-repeat microtubule-binding domain (MBD) in the tau protein, four two-repeat peptides (R12, R13, R23 and R34) and two three-repeat peptides (R123 and R234) were prepared, and their in vitro self-aggregation was investigated by thioflavin S fluorescence and circular dichroism measurements, and by electron microscopy in neutral buffer (pH 7.6). Comparison of these aggregation behaviors with previous results for single-repeat peptides and wild-type 3RMBD (R134) and 4RMBD (R1234) indicated that (a) the two-repeat R23, not the R2 or R3 single repeat, forms the core structure in self-aggregation of 4RMBD, whereas that of 3RMBD comprises the R3 single repeat, (b) co-existence of R1 and R4 repeats is necessary for the aggregation behavior inherent in 3RMBD and 4RMBD, whereas the R1 or R4 repeat alone functions as a repressor or modifier of the filament formation, (c) 4RMBD aggregation is accompanied by R1-driven transition from random and alpha-helix structures to a beta-sheet structure, whereas 3RMBD aggregation involves three-repeat R134-specific transition from a random structure to an alpha-helix structure without the participation of a beta-sheet structure, and (d) the peptides that include the R1 repeat form a long filament irrespective of the absence or presence of the R4 repeat, whereas those that include the R4 repeat, but not the R1 repeat, form a relatively short filament. To the best of our knowledge, a systematic study of the linkage-dependent contribution of each repeat peptide to the paired helical filament formation of tau MBD has not been carried out previously, and thus the present information is useful for understanding the essence of the filament formation of tau MBD.

Heparan sulfate and heparin enhance ERK phosphorylation and mediate preBCR-dependent events during B lymphopoiesis.

As B lineage cells develop, they interact with cells, proteins, and extracellular matrix components of the surrounding microenvironment. In vitro, one critical checkpoint for developing cells occurs as they lose responsiveness to IL-7. These cells require contact with either stromal cells or other B lineage cells to mature. Our results demonstrate that heparan sulfate and heparin are able to promote this transition when added exogenously to the culture system or when heparan sulfate-bearing cell lines are cocultured with primary B cell progenitors. Addition of heparan sulfate or heparin to LPS-stimulated cultures of primary B cell progenitors resulted in more IgM secreted compared with untreated cultures. Heparan sulfate has been reported to be a ligand for the pre-B cell receptor (preBCR). Extending this observation, we found that treatment of preBCR+ cells with heparan sulfate before anti-micro stimulation leads to increased phosphorylation of ERK1/2. Consequently, preBCR+ cells proliferate more in the presence of IL-7 and heparan sulfate, whereas preBCR- cells are unaffected, suggesting that in these experiments, heparan sulfate is not directly affecting IL-7 activity. Heparin treatment of cultures induces many of the same biological effects as treatment with heparan sulfate, including elevated pERK levels in preBCR+ cells. However, heparin reduces the proliferation of cells expressing only the preBCR (opposed to both the preBCR and BCR) possibly due to internalization of the preBCR. Heparan sulfates are present on stromal cells and B lineage cells present in hemopoietic tissues and may provide stimulation to preB cells testing the signaling capacity of the preBCR.